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論文名稱(外文):Biochemical and structural studies on CARD9
指導教授(外文):Yu-Chih Lo
外文關鍵詞:CARD9BCL10CBM complex
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When cell receives external stimuli, CARMA family recruits downstream components BCL10 as well as MALT1 to form a signaling complex called the CBM complex. The CBM complex induces NF-B signaling pathway against pathogen invasion. CARD9 is structurally dissimilar to other members in CARMA family, which lacks MAGUK domain for maintaining inactive state. Previous research showed that CARD9 involved in the antifungal, antibacterial as well as antiviral immune responses, and CARD9 deficiency leads to immune system disorders and fungal infections. It shows that CARD9 plays a crucial role in immune system. However, mechanism of how CARD9 forms the CBM complex with BCL10 and MALT1 is still unknown. Hence, I propose to solve the structure of CARD9 and use biochemical assays to reveal its molecular mechanism in NF-B pathway. Different lengths of CARD9 are constructed via secondary structure prediction and purified by the affinity chromatography and size exclusion chromatography. The pure and large quantity of CARD9 observed under electron microscope via the negative stain showed high contrast between the particle of CARD9 and background, which contributed to data collection and calculation. Besides, CARD9 co-purified with different lengths of BCL10 could form complex with BCL10. Furthermore, the result of the pull down assay showed that the key mutants on BCL10 enhanced binding affinity with CARD9, suggested that the protein complex could be further obtained. The result got from above experiments, it will help us to know the structural information and molecular characterization of CARD9 in NF-B pathway.
Chinese Abstract (中文摘要).....I
Table of Contents.....VI
Contents of Tables.....VIII
Contents of Figures.....IX
Contents of Appendices.....XI
Abbreviation List.....XII
1.Research Background.....1
1-1 The NF-κB signaling pathway.....1
1-2 Caspase recruitment domain (CARD) and membrane-associated guanylate kinase-like domain (MAGUK) protein, CARMA family.....2
1-3 Caspase recruitment domain-containing protein 9 (CARD9).....4
1-4 The aim of this study.....5
2.Materials and Methods.....7
2-1 The preparation of competent cell.....7
2-2 Reconstruction of CARD9.....7
2-3 Mutagenesis of CARD9.....8
2-4 Protein expression constructs.....9
2-5 Recombinant protein expression and purification.....10
2-6 X-ray crystallography.....10
2-7 Negative stain electron microscopy (NS-EM).....11
2-8 Multiangle light scattering (MALS).....12
2-9 Pull down assay of CARD9 and BCL10.....12
3-1 Expression and purification of recombinant proteins.....13
3-2 The negative staining results of different lengths of CARD9 protein.....16
3-3 X-ray crystallography.....17
3-4 Multi Angle Light Scattering (MALS).....18
3-5 Co-purification of CARD9 and BCL10 to form complex.....19
3-6 CARD9 pulldown BCL10 mutants.....21
3-7 Mutagenesis CARD9 and pulldown BCL10 mutants.....22
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